EP1384777A1 - Adn genomiques participant a la polyarthrite rhumatoide, son procede de diagnostic, son procede d'estimation du risque d'apparition et trousse de diagnostic de detection associee - Google Patents

Adn genomiques participant a la polyarthrite rhumatoide, son procede de diagnostic, son procede d'estimation du risque d'apparition et trousse de diagnostic de detection associee Download PDF

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Publication number
EP1384777A1
EP1384777A1 EP02713262A EP02713262A EP1384777A1 EP 1384777 A1 EP1384777 A1 EP 1384777A1 EP 02713262 A EP02713262 A EP 02713262A EP 02713262 A EP02713262 A EP 02713262A EP 1384777 A1 EP1384777 A1 EP 1384777A1
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Prior art keywords
rheumatoid arthritis
same
genomic dna
diagnosing
detecting
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EP02713262A
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German (de)
English (en)
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EP1384777A4 (fr
EP1384777B1 (fr
Inventor
Shunichi Shiozawa
Koichiro Komai
Hirofumi Yagi
Nao Matsuura
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to genomic DNAs with mutations, a method of diagnosing human rheumatoid arthritis by using the mutations, a method of judging onset risk thereof, and a diagnostic kit for detecting the same.
  • RA Rheumatoid arthritis
  • RA Rheumatoid arthritis
  • Untreated RA causes a destruction and a deformation of joint, later presenting functional disorders of motor apparatus. Sometimes it threatens lives of patients. Consequently, patients with RA have to bear large, lifelong physical and mental burdens.
  • RA results in a large variety of symptoms, and the diagnostic criteria of the American College of Rheumatology are widely used for its diagnosis.
  • development of an onset state of RA is generally very slow, requiring a period from several weeks to several months.
  • rheumatoid factor which is an objective index in the diagnostic criteria of American College of Rheumatology
  • a positive rate is 33% within 3 months and around 88% even after 12 months or more [Chiryo, 73(3): 23-27, 1991]. This indicates that RA cannot be diagnosed definitively at present.
  • An attempt to diagnose rheumatoid arthritis by detecting a serum rheumatoid arthritis associated antigen IgM in a patient through a reaction with recombinant antigen has been performed (JP-A-10-513257).
  • a therapeutic procedure to be selected is generally varied depending on a progression stage of symptoms in the disease state.
  • a nonsteroidal antiinflammatory drug NSAID
  • a disease-modifying antirheumatic drug DMARD
  • the NSAID is administered, and at the same time, an effort is made to identify this disease from other rheumatic diseases including collagen disease, by carefully observing the symptom and procession.
  • steroids may be administered, and a pharmacotherapy for pain together with a physiotherapy and an orthotic therapy are performed in order to maintain and ameliorate joint functions.
  • a surgical therapy may be performed in a case in which daily life is inconvenienced by a joint disruption.
  • RA is still thought to be a disease which develops and progresses after an onset caused by cooperation with a large number of causative factors including a living environment. For that reason, in order to perform a more exact elucidation of the disease and a proper therapy thereof, an essential part of interactions of the multiple factors involved has to be established. Since RA is a disease with an incident rate of 1% or less in the world (N. Engl. J.
  • An object of the present invention is to elucidate further mutations in human Dbl gene and their relations to an onset or an onset risk of RA; and provides a method for precisely diagnosing the onset or the onset risk of RA by utilizing such mutations.
  • Another object of the present invention is to provide a diagnostic kit useful for detecting a genomic DNA which is a mutated Db1 gene associating with RA.
  • the base (c) at the position-1987 is located in the intron 24, and the bases (t) and (a) at the position-3664 and the position-3769 are located in the intron 23.
  • the mutated genomic DNA of the present invention is now known to be partially the cause of the RA onset.
  • Such a relationship between single base substitution mutation and diseases is known in other cases such as the gene causative of type II diabetes mellitus (Nature Genetics, 26: 163-175, 2000).
  • the present inventors have found from these studies that a method of diagnosing RA, a method of judging the onset risk of RA, and a diagnostic kit for detecting these mutations, by using the mutations of the Dbl gene in cells obtained from examinees as a index, are useful, and accomplished the present invention. Further, the present invention is useful for developments of novel preventive or therapeutic methods and drugs for the treatment of rheumatoid arthritis.
  • a, c, g and t mean the bases adenine, cytosine, guanine and thymine, respectively.
  • SEQ ID NO: 1 corresponds to the sequence from the position-55,823 to the position-59,696 of the sequence registered in GenBank as the human genome of the X chromosome, q25-26.3 region containing the genomic DNA of the Dbl gene (GenBank accession No. AL033403).
  • the sequence registered in GenBank has the following properties: The complementary strand thereof is a +strand; it is translated from the position-115,837 to 5' direction; and it transcribes mRNA of the GenBank accession No. X12556.
  • the method of diagnosing RA, the method of judging onset risk thereof, and the diagnostic kit for detecting the same in the present invention detects at least one of the mutations in the genomic DNA previously described.
  • Identification of a mutant genomic DNA and diagnosis of RA or judgment of onset risk of RA can be made, for example, as described below.
  • a genomic DNA of an examinee can be obtained by the conventional method from any human cells, for example, hair, various organs, peripheral lymphocytes and synovial cells. It can also be obtained from cultured and proliferated cells. In addition, the thus obtained genomic DNA can be used after being amplified by using the conventional gene amplification methods such as PCR (Polymerase chain reaction), NASBA (Nucleic acid sequence based amplification), TMA (Transcription-mediated amplification) and SDA (Strand displacement amplification).
  • PCR Polymerase chain reaction
  • NASBA Nucleic acid sequence based amplification
  • TMA Transcription-mediated amplification
  • SDA String displacement amplification
  • Detection method for genomic variants is not particularly limited. It includes, for example, allele specific oligonucleotide probe method, oligonucleotide ligation assay method, PCR-SSCP method, PCR-CFLP method, PCR-PHFA method, invader method, RCA (Rolling circle amplification) method and primer oligo base extension method.
  • a PCR primer which can amplify the region containing the mutated positions in SEQ ID NO: 1, is synthesized; and then direct sequencing of the PCR product amplified from a genomic DNA of the examinee is carried out to determine the mutation.
  • diagnosis of RA of the examinee or onset risk thereof can then be judged precisely.
  • the primer used in the present invention can be prepared conventionally by using a DNA synthesizer or the like.
  • the mutations described above can also be detected by using a microarray equipped with oligonucleotides consisting of a normal sequence and a mutated sequence in the mutation site penumbra.
  • the mutation (t ⁇ g) at the position-3664 can also be detected, as shown in the Examples, by amplifying a +strand of the genomic DNA using a synthetic oligonucleotide containing the mutation site thereof (SEQ ID NO: 2 and SEQ ID NO: 3) as a primer in PCR, cleaving the PCR product with the restriction enzyme Hinf-I, and examining whether the PCR product is fragmented into two fragments or not (RFLP analysis).
  • the normal sequence penumbral to the position-3664 in the complementary strand of SEQ ID NO: 1 is 5'-gaatc-3', and is cleaved by Hinf-I (recognition sequence: 5'-g ⁇ antc-3').
  • the mutated sequence penumbral to the position-3664 in the complementary strand of SE ID NO: 1 is 5'-gcatc-3', and is not cleaved by Hinf-I.
  • a diagnostic kit of the present invention is not particularly limited as long as it contains a reagent such as primer and probe, which can detect at least one mutation of the genomic DNA described above, and can be obtained by further combining other additional reagents.
  • kits examples include a combination of a primer which is designed to amplify the genomic region containing at least one mutation described above, and further at least one reagent necessary for detecting the mutation including a prove which is designed to detect the genomic region containing at least one mutation described above, a restriction enzyme and a reagent used for base sequence determination methods such as Maxam-Gilbert method and chain termination method.
  • a kit comprising a fluorescence labeled dideoxynucleotide is further included.
  • Diagnosis of RA or onset risk thereof can be performed precisely by using the diagnostic kit.
  • the diagnostic kit of the present invention can be constructed, for example, in the case of a kit for RFLP analysis of the mutation at the position-3664, by a primer set consisting of base sequences of SEQ ID NO: 2 and SEQ ID NO: 3, restriction enzyme Hinf-I, DNA synthase, and the like. Further, proper buffer, washing solution and the like, which do not disturb the detection of mutation, may be added.
  • Genomic DNA was extracted from the peripheral blood. After amplifying the region with about 5.3 kbp penumbral to the exon 23 and 24 which correspond to 223 bp deficient region of Dbl gene cDNA by PCR, the base sequence was determined by Dye Terminator method using a sequence primer (Fig. 1) which was designed based on the previously known genomic sequence (Acc. No. AL033403.1). In the statistical analysis, chi-square test ( ⁇ 2 -test) by the percentage method was used for the test of significance. Base sequence of each primer set used for the PCR is shown as follows:
  • nt2632+211(a ⁇ c) corresponds to t ⁇ g mutation at the position-3664 in SEQ ID NO: 1
  • nt2745+576(g ⁇ a) corresponds to c ⁇ t mutation at the position-1987 in SEQ ID NO: 1.
  • Genomic DNA 1 ⁇ l PCR buffer II (Applied Biosystems Inc.) 2.5 25 mM MgCl 2 1.5 2 mM dNTP 2.5 10 pmol/ ⁇ l sense primer 0.5 10 pmol/ ⁇ l antisense primer 0.5 Gold Taq polymerase 0.25 Sterilized water 16.25
  • DNA-amplified reaction mixture was reacted at 37°C for 1 hour with a restriction enzyme Hinf-I (New England Biolabs Inc., recognition sequence: 5'-G ⁇ ANTC-3') using the reaction composition described below to digest completely, and analyzed by the conventional manner using 2.0% agarose gel electrophoresis and ethidium bromide staining.
  • PCR reaction mixture 10 ⁇ l Hinf-I 2 ⁇ l Reaction buffer (NE Buffer II) 1.5 ⁇ l Sterilized water 1.5 ⁇ l
  • Lane 2 is an electrophoretic pattern of the PCR product after the Hinf-I treatment obtained by using the normal Dbl gene as a template wherein the sequence is cleaved to 227 bp and 144 bp by the Hinf-I recognition sequence (5'-gaatc-3') at the position-3664 (nt2632+211) penumbra.
  • Lane 1 is an electrophoretic pattern of the PCR product derived from the homologous variant Dbl gene wherein no cleavage occurs due to disappearance of the Hinf-I recognition sequence by t ⁇ g mutation at the position-3664 (a ⁇ c mutation of nt2632+211).
  • Lane 3 is an electrophoretic pattern of the PCR product derived from the heterologous variant Dbl gene. Since a fragment having the Hinf-I recognition sequence and a fragment without having the Hinf-I recognition sequence were amplified, 3 fragments consisting of a non-cleaved fragment of 377 bp and further cleaved 2 fragments of 227 bp and 144 bp were simultaneously detected.
  • the present invention relates to genomic DNAs with mutations associated with human rheumatoid arthritis, a method of diagnosing human rheumatoid arthritis by using these mutations, a method of judging onset risk thereof, and a diagnostic kit for detecting the same.
  • the present invention is useful for detecting onset of rheumatoid arthritis or onset risk thereof precisely, simply and exactly. Further, the present invention is useful for developing novel preventive and therapeutic methods and therapeutic drugs for rheumatoid arthritis.

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  • Life Sciences & Earth Sciences (AREA)
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EP02713262A 2001-03-30 2002-03-29 Adn genomiques participant a la polyarthrite rhumatoide, son procede de diagnostic, son procede d'estimation du risque d'apparition et trousse de diagnostic de detection associee Expired - Lifetime EP1384777B1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP2001102006 2001-03-30
JP2001102006 2001-03-30
PCT/JP2002/003191 WO2002079466A1 (fr) 2001-03-30 2002-03-29 Adn genomiques participant a la polyarthrite rhumatoide, son procede de diagnostic, son procede d'estimation du risque d'apparition et trousse de diagnostic de detection associee

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EP1384777A1 true EP1384777A1 (fr) 2004-01-28
EP1384777A4 EP1384777A4 (fr) 2005-06-01
EP1384777B1 EP1384777B1 (fr) 2009-05-27

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US (1) US7514211B2 (fr)
EP (1) EP1384777B1 (fr)
JP (1) JPWO2002079466A1 (fr)
KR (1) KR100890448B1 (fr)
AT (1) ATE432345T1 (fr)
AU (1) AU2002244949B2 (fr)
CA (1) CA2443146C (fr)
DE (1) DE60232442D1 (fr)
DK (1) DK1384777T3 (fr)
ES (1) ES2325807T3 (fr)
PT (1) PT1384777E (fr)
WO (1) WO2002079466A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1008648A1 (fr) * 1997-05-15 2000-06-14 Shunichi Shiozawa Gene responsable de l'arthrite rhumatoide, methode pour diagnostiquer l'arthrite rhumatoide et procede permettant de determiner des facteurs responsables de l'arthrite rhumatoide
EP1164190A1 (fr) * 1999-03-20 2001-12-19 Shunichi Shiozawa Gene de la polyarthrite rhumatoide et procede permettant de diagnostiquer la polyarthrite rhumatoide

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6582908B2 (en) * 1990-12-06 2003-06-24 Affymetrix, Inc. Oligonucleotides
AU5052999A (en) * 1998-07-25 2000-02-21 Astrazeneca Limited Human ccr-2 gene polymorphisms
AU5876899A (en) 1998-09-19 2000-04-10 Astrazeneca Ab Polymorphisms in the human alpha4 integrin subunit gene, suitable for diagnosis and treatment of integrin ligand mediated diseases

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1008648A1 (fr) * 1997-05-15 2000-06-14 Shunichi Shiozawa Gene responsable de l'arthrite rhumatoide, methode pour diagnostiquer l'arthrite rhumatoide et procede permettant de determiner des facteurs responsables de l'arthrite rhumatoide
EP1164190A1 (fr) * 1999-03-20 2001-12-19 Shunichi Shiozawa Gene de la polyarthrite rhumatoide et procede permettant de diagnostiquer la polyarthrite rhumatoide

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO02079466A1 *

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Publication number Publication date
WO2002079466A1 (fr) 2002-10-10
US20040175706A1 (en) 2004-09-09
ATE432345T1 (de) 2009-06-15
CA2443146C (fr) 2011-03-29
JPWO2002079466A1 (ja) 2004-07-22
EP1384777A4 (fr) 2005-06-01
US7514211B2 (en) 2009-04-07
KR100890448B1 (ko) 2009-03-26
CA2443146A1 (fr) 2002-10-10
DK1384777T3 (da) 2009-09-07
EP1384777B1 (fr) 2009-05-27
PT1384777E (pt) 2009-06-12
DE60232442D1 (de) 2009-07-09
KR20030093291A (ko) 2003-12-06
ES2325807T3 (es) 2009-09-18
AU2002244949B2 (en) 2007-08-30

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